The present invention relates to an improved method and kit for direct radiolabeling of a monovalent antibody fragment with a radiometal ion of a radionuclide that binds to sulfhydryl groups, using one or more pendant sulfhydryl groups as endogenous ligands and optionally using an exogenous ligand which further stabilizes the bound radiometal ion. More particularly, the invention relates to a method and kit for direct radiolabeling of Fab or Fab' antibody fragments.
The present invention provides certain improvements over the general methods disclosed in Shochat et al., U.S. patent application Ser. No. 07/176,421, filed Apr. 1, 1988, now U.S. Pat. No. 5,061,641.
It is known that certain radiometals display affinities for sulfur ligands, including, e.g., Tc-99m from reduced pertechnetate, Re-186 and Re-188 ions, Cu-67 ions, Hg-197 ions and Bi-212 ions. Some of these radiometals have been bound to proteins, especially antibodies or antibody fragments, using conjugated chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or a variety of sulfur/nitrogen (S.sub.2 N.sub.2) chelators such as bis-thiosemicarbazones and the like.
Methods have been reported for binding Tc-99m ions directly to antibodies by pre-tinning the protein and then contacting the resultant material with pertechnetate. This procedure often does not work well and some of the radiometal is bound to sites which are comparatively labile in the presence of blood and other bodily fluids or tissues. The mechanism of the pre-tinning process is not well understood and the reasons for production of labile sites of labeling have not been elucidated.
It is also known that proteins containing disulfide bonds can be reduced to produce pendant sulfhydryl groups. If the disulfide bonds link polypeptide chains which are not themselves joined, e.g., antibody light/heavy chains, reductive cleavage thereof can dissociate the protein into smaller fragments. An example of this is the reductive cleavage of antibody F(ab').sub.2 fragments to form Fab' fragments using disulfide reducing agents such as cysteine, dithiothreitol, mercaptoethanol and the like.
Direct labeling of a protein has the advantage that chelate conjugates are obviated. However, attempts to use such radiolabeled proteins for in vivo applications, e.g., antibody-targeted tumor imaging and therapy, have revealed problems due to loss of the label to other organs which retain it and cause both high background for certain types of imaging and slow clearance of the label from organs such as the liver, spleen and kidneys. The latter problem can in turn cause hepatic and/or renal crisis for the therapy patient due to severe compromise of the affected organ by the high levels of radiation.
It has been discovered that the presence of certain disulfide reducing agents having free -SH groups (e.g. cysteine) can interfere with subsequent labeling, due to binding of the radionuclide cations to the free -SH groups of the reducing agents. In such cases a purification step is required to remove the reducing agent before the product is labeled.
A need exists for a direct method for radiolabeling a monovalent antibody fragment that produces a good yield of a stable radiolabeled product which retains the label in the presence of blood and other body fluids and tissues, and which does not require a purification step to remove a disulfide reducing agent.